CN116059352A - Application of human annexin ANX1 in preparation of inflammatory disease medicines - Google Patents
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- CN116059352A CN116059352A CN202211508684.7A CN202211508684A CN116059352A CN 116059352 A CN116059352 A CN 116059352A CN 202211508684 A CN202211508684 A CN 202211508684A CN 116059352 A CN116059352 A CN 116059352A
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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Abstract
The invention discloses an application of human annexin ANX1 in preparing medicines for inflammatory diseases. The human ANX1 has the ability to inhibit Th17 cell differentiation and treatment of ulcerative colitis. The human ANX1 has the capability of inhibiting the differentiation of Th17 cells, and in an autoimmune colitis mouse model, the human recombinant human ANX1 plays a role in immunosuppression by regulating SOCS3/STAT3 signaling and limiting the development of Th17, so that the severity of inflammatory diseases is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of human annexin ANX1 in preparation of inflammatory disease medicines.
Background
Ulcerative colitis (ulcerative colitis, UC) is a recurrent autoimmune disease, which is one of the inflammatory bowel diseases (Inflammatory bowel disease, IBD). The typical clinical symptoms of ulcerative colitis are bloody diarrhea, nocturnal bowel movement, emergency and tenesmus, and after the initial occult period, it is characterized by recurrent episodes and remission of mucosal inflammation. The pathogenesis of IBD is not yet defined, and genetic, immunological and environmental factors such as diet, stress, smoking and free radicals are involved in its development.
Worldwide, the incidence of UC has a rising trend year by year. The incidence of UC is high in developed countries, whereas by the beginning of the 21 st century, the incidence of UC has tended to stabilize in developed countries and has risen in many new industrialized countries in south america, asia, africa and the middle east. Ulcerative colitis has been very rare in China, the total incidence rate is 1.2/10 ten thousand people/year, but the ulcerative colitis has a rapid rising trend along with the development of economy in China. With the rising prevalence of UC, overall medical costs will also rise. Therefore, development of novel therapeutic agents with high efficiency and low toxicity is demanded.
anti-TNF-alpha antibody drugs were developed in the late 90 s of the 20 th century, and have achieved great success in the treatment of autoimmune diseases such as inflammatory bowel disease, ankylosing spondylitis, psoriasis and rheumatoid arthritis. Currently common TNF-alpha antibody drugs include infliximab, adalimumab, golimumab, cetuzumab, and etanercept.
Although TNF-a antibody drugs have achieved a remarkable effect in autoimmune diseases, reports on adverse reactions are increasing with the efficient treatment of the above diseases. IBD, ankylosing spondylitis and rheumatoid arthritis patients (20% -22%) developed new forms of psoriasis after treatment with anti-TNF- α antibody drugs (Puig L et al, dermatology 2012,225 (1): 14-17; gannes G et al, arch. Dermatol.2007,143 (2): 223-231; koJM et al, J. Dermatol. Treat 2009, 20 (2): 100-108). This adverse reaction developed markedly in responders rather than non-responders and led to 5% to 35% of patients discontinuing anti-TNF-alpha therapy (Rahier J et al, clin. Gastreantol. Hepatol.2010,8 (12): 1048-1055;Scaldaferri F et al, glut 2014,63 (4): 699-701). However, in contrast to rheumatoid arthritis and IBD patients, the cumulative dose had no effect on the time to progression of psoriasis (Scaldaferri F et al, gut 2014,63 (4): 699-701). Furthermore, one study showed that skin lesions of IBD patients receiving anti-TNF- α treatment were infiltrated by Th17 cells (tillock C et al, gut 2014,63 (4): 567-577).
Th17 cells are a subset of T cells characterized by the production of large amounts of IL-17A, IL-17F, IL-21 and IL-22.Th17 cells are differentiated under co-induction by IL-6 and TGF-beta, and expansion thereof is promoted by IL-23 (Caprioli F et al, J. Crohn's Colitis 2008,2 (4): 291-295). IL-21 produced by Th17 cells in turn increases the expression of its IL-23 receptor, thereby creating a positive autoregulatory feedback loop that enhances expansion of Th17 cells. The role of Th17 cells, and in particular of the secreted characteristic cytokine IL-17A, in intestinal inflammation has been widely studied. The secretion and mRNA levels of IL-17A in the serum and diseased intestinal mucosal tissues of IBD patients are significantly higher than normal. The severity of UC disease correlated positively with the amount of IL-17A expressed, indicating that IL-17A in the intestine promoted the progression of disease in IBD patients (Gannes G et al, arch. Dermatol.2007,143 (2): 223-231; curlen G et al, aliment. Pharmacol. Ther.2011,34 (11-12): 1318-1327; milanez FM et al, arthritis Res. Therapy 2016,18 (1): 52). At present, induction of Th17 cells in the intestinal tract has been studied under various conditions and factors. It has been shown that antigen-specific Th17 differentiation occurs in the Lamina Propria (LP) of the gut, and that cd11c+ DC cells can treat antigens such as microorganisms by phagocytosis or pinocytosis, stimulating Th17 production via the mhc ii pathway, and this process is independent of mesenteric lymph nodes and gut-associated lymphoid tissue.
Annexin A1 (ANX 1) originates from a process that investigates the mechanism by which glucocorticoids inhibit prostaglandin synthesis, also known as large corticoids, renal corticoids, fat regulatory proteins and lipocortin 1, characterized by inhibiting eicosanoid production by affecting the activity of phospholipase A2 (PLA 2). ANX1 is a monomeric amphiphilic protein consisting of 346 amino acids, which is one of the annexin superfamily and has a size of 37kDa. ANX1 is expressed predominantly in subcellular particles of neutrophils, eosinophils and monocytes, with minor expression in specific lymphocyte subsets.
Recent evidence suggests that ANX1 mediates most of its cellular effects through interactions with formyl peptide receptors (Formyl peptide receptor, FPR). Formyl peptide receptors are members of a family of G protein-coupled receptors comprising seven transmembrane domains, mainly expressed in mammalian phagocytic leukocytes, which induce responses to various ligands, playing an important role in host defense and inflammation. There are three FPR receptors in humans, namely FPR1, FPR2 and FPR3, and the three FPRs have significant sequence homology.
Secretion of ANX1 was detected in inflammatory colon biopsies of severe UC patients, whereas it was not detected in biopsies of healthy human colon, non-UC patients or of mild or moderate UC patients. Endogenous ANX1 plays a role in anti-TNF treatment of DSS acute colitis mice models, infliximab can prevent clinical symptoms of DSS-induced WT colitis, but cannot be used in ANX 1-/-mice (Gaffen SL et al, nat. Rev. Immunol.2009,9 (8): 556), suggesting that TNF- α antibody drug infliximab needs to exert an acute colitis treatment effect through endogenous ANX1. However, if ANX1 is used as an exogenous drug, it acts outside the cell but cannot enter the cell, how ANX1 acts and how it acts with TNF- α antibody drugs.
At present, the application of human annexin ANX1 in preparing medicines for inflammatory diseases is lacking.
Disclosure of Invention
The invention aims at solving the defect of the existing TNF-alpha antibody for treating ulcerative colitis, and provides an application of annexin ANX1 in preparation of ulcerative colitis medicines.
In order to solve the problems, the invention provides the following technical scheme: the invention relates to an application of human annexin ANX1 in preparing medicines for inflammatory diseases.
Further, the inflammatory disease is ulcerative colitis.
Further, the human ANX1 has the capability of treating ulcerative colitis, and the human ANX1 is shown as SEQ ID No. 1.
Further, the human ANX1 has the ability to inhibit Th17 cell differentiation.
Furthermore, in the mouse model of autoimmune colitis, recombinant human ANX1 exerts its immunosuppressive effects by modulating SOCS3/STAT3 signaling, limiting TH17 development, thus reducing the severity of inflammatory disease.
Furthermore, recombinant human ANX1 exerts its immunosuppressive or inflammation-suppressing effects by regulating SOCS3/STAT3 signaling, limiting Th17 cell development, and inhibiting Th17 cell differentiation.
The preparation method of the human annexin ANX1 comprises the following steps: (1) The human ANX1 gene shown as SEQ ID No.1 is introduced into a pET28a vector through enzyme digestion and enzyme linked reaction to construct an expression plasmid;
(2) The target plasmid is transferred into BL21 (DE 3) competent cells, and after transformation, a proper amount of bacterial liquid is coated on LB plate containing kanamycin for overnight culture at 37 ℃.
(3) Positive clones were picked and grown in LB medium containing kanamycin and induced by addition of 5mM IPTG at 15℃for 20h. The cell pellet was collected by centrifugation, protein buffer was added to the system, the cells were broken using a homogenizer, and the supernatant was collected after centrifugation. Separating and purifying by column chromatography, and specifically performing steps such as TNF-alpha antibody purification to obtain recombinant human annexin ANX1.
The beneficial effects are that: the human ANX1 has the capability of inhibiting Th17 cell differentiation, and exogenous recombinant human ANX1 plays a role in immunosuppression by regulating SOCS3/STAT3 signaling and limiting development of Th17 in an autoimmune colitis mouse model, so that the severity of inflammatory diseases is reduced.
Drawings
FIG. 1 is a diagram of the construction of the ΔCIITA-RAW264.7 of the present invention; a: detecting down-regulation of MHCII in ΔCIITA-CPR-2 by qRT-PCR; b: TGF- β and IL-6 were down-regulated in ΔCIITA-CPR-2 as detected by qRT-PCR.
FIG. 2 is a graph of the effect of ΔCIITA-CPR of the present invention on Th 17; a: deltaCIITA-CPR-2 induces a streaming analysis of Th17 in the colon; b: deltaCIITA-CPR-2 induced a streaming analysis of Th17 in mLN.
FIG. 3 is a graph of ANX1 in the colon significantly upregulated by CPR-2 of the TNF-alpha antibody-TNF-alpha antigen/antibody complex of the present invention; a: by qRT-PCR analysis, expression of ANX1 in ΔCIITA-CPR-2 was down-regulated compared to TNF-alpha antibody-TNF-alpha antigen/antibody complex CPR-2; b: flow cytometry analysis mLN and expression of ANX1 binding receptors in the colon.
FIG. 4 is a graph of ANX1 activation FPR2 of the present invention; a: qRT-PCR detects the expression level of ANX1 after CPR stimulation of colon LPL; b: WRW4 (10. Mu.g/ml) inhibited the expression of FPR 2.
FIG. 5 is a graph showing the effect of different doses of ANX1 according to the invention on the body weight and colon length of DSS mice; a: ANX1 treats a range of body weight changes during DSS-induced colitis; b: representative colon length comparisons for each group on day 8.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
As shown in fig. 1 to 5, the use of human annexin ANX1 according to the invention for the preparation of a medicament for inflammatory diseases including ulcerative colitis.
The ANX1 has the capability of treating ulcerative colitis, and the ANX1 is shown as SEQ ID No. 1.
The ANX1 has the ability to inhibit Th17 cell differentiation. In the mouse model of autoimmune colitis, human recombinant ANX1 exerts its immunosuppressive effects by regulating SOCS3/STAT3 signaling, limiting TH17 development, thus reducing the severity of inflammatory disease.
Example 2
The preparation method of the human ANX1 comprises the following steps:
the human ANX1 gene shown as SEQ ID No.1 is introduced into a pET28a vector through enzyme digestion and enzyme linked reaction to construct an expression plasmid;
the target plasmid is transferred into BL21 (DE 3) competent cells, and after transformation, a proper amount of bacterial liquid is coated on LB plate containing kanamycin for overnight culture at 37 ℃.
Positive clones were picked and grown in LB medium containing kanamycin and induced by addition of 5mM IPTG at 15℃for 20h. The cell pellet was collected by centrifugation, protein buffer was added to the system, the cells were broken using a homogenizer, and the supernatant was collected after centrifugation. Separating and purifying by column chromatography, and specifically performing steps such as TNF-alpha antibody purification to obtain recombinant human ANX1 protein.
Test example 1
The invention proves that ANX1 has a therapeutic effect on DSS-induced colonitis, and the specific steps are as follows:
model induction was performed in 7-9 week C57BL/6 female mice (body weight 18-20 g). Mice in the blank control group were fed normal drinking water daily, and mice in the DSS manufacturing module and each dosing treatment group were free to drink drinking water containing 3% DSS for a total of 8 days for induction.
Starting from the second day of molding, mice were intraperitoneally injected with human ANX1. Mu.g/kg, 500. Mu.g/kg, 2mg/kg, and the dosing period was once every three days.
The body weight of the mice and fecal occult blood were recorded daily.
As shown in FIG. 5, the colon length and body weight of the 500 μg/kg group were closer to those of the control group and significantly higher than those of the DSS group, indicating that at a dose of 500 μg/kg, human ANX1 had a therapeutic effect on DSS-induced colitis, which was the optimal therapeutic dose.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. An application of human annexin ANX1 in preparing medicines for treating inflammatory diseases.
2. The use according to claim 1, characterized in that: the inflammatory disease is ulcerative colitis.
3. The use according to claim 1, characterized in that: the amino acid sequence of the human annexin ANX1 is shown as SEQ ID No.1, and the human annexin ANX has the capability of inhibiting Th17 cell differentiation and treating ulcerative colitis.
4. A use according to claim 3, characterized in that: the human annexin ANX1 has the capacity of inhibiting Th17 cell differentiation.
5. The use according to claim 4, characterized in that: in the mouse model of autoimmune colitis, recombinant human ANX1 exerts its immunosuppressive effects by regulating SOCS3/STAT3 signaling, limiting Th17 cell development, thus reducing the severity of inflammatory disease.
6. The use according to claim 5, characterized in that: recombinant human ANX1 exerts its immunosuppressive or inflammation-suppressing effects by regulating SOCS3/STAT3 signaling, restricting Th17 cell development, and inhibiting Th17 cell differentiation.
7. Method for the preparation of ANX1 according to any one of claims 1 to 6, characterized in that it comprises the following steps: (1) Introducing an ANX1 gene shown as SEQ ID No.1 into a pET28a vector through enzyme digestion and enzyme linked reaction to construct an expression plasmid;
(2) Transferring the target plasmid into BL21 competent cells, and after transformation, taking a proper amount of bacterial liquid, coating the bacterial liquid on an LB plate containing kanamycin, and culturing overnight at 37 ℃;
(3) Positive clones were picked and expanded in LB medium containing kanamycin, and induced by adding 5mM IPTG at 15℃for 20h; collecting cell sediment by centrifugation, adding protein buffer solution into the system, crushing cells by using a homogenizer, and collecting supernatant after centrifugation; separating and purifying by column chromatography to obtain recombinant human annexin ANX1.
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| CN202211499337.2A Pending CN117257933A (en) | 2022-03-18 | 2022-11-28 | Preparation method and application of TNF-alpha nano antibody |
| CN202211499338.7A Pending CN116173200A (en) | 2022-03-18 | 2022-11-28 | TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines |
| CN202211508681.3A Pending CN115779080A (en) | 2022-03-18 | 2022-11-29 | Application of annexin ANX1 and TNF-alpha nano antibody combination in preparation of ulcerative colitis medicine |
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| EP1724586A2 (en) * | 2005-05-21 | 2006-11-22 | ProteoSys AG | Annexin for cancer risk assessment |
| CN102272156A (en) * | 2008-12-02 | 2011-12-07 | 玛丽皇后和威斯特-弗尔德学院 | Treatment of autoimmune disease by regulating annexin-1 |
| CN106497956A (en) * | 2016-10-25 | 2017-03-15 | 上海交通大学 | CYP101 enzymes recombinant vector and construction method, CYP101 enzyme high efficient expression purification process |
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|---|---|---|---|---|
| EP1724586A2 (en) * | 2005-05-21 | 2006-11-22 | ProteoSys AG | Annexin for cancer risk assessment |
| CN102272156A (en) * | 2008-12-02 | 2011-12-07 | 玛丽皇后和威斯特-弗尔德学院 | Treatment of autoimmune disease by regulating annexin-1 |
| CN106497956A (en) * | 2016-10-25 | 2017-03-15 | 上海交通大学 | CYP101 enzymes recombinant vector and construction method, CYP101 enzyme high efficient expression purification process |
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| XING LIU等: "Exogenous Annexin 1 inhibits Th17 cell differentiation induced by anti-TNF treatment via activating FPR2 in DSS-induced colitis", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 107, 17 March 2022 (2022-03-17), pages 108685, XP087044770, DOI: 10.1016/j.intimp.2022.108685 * |
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